Diabetes mellitus is a disease caused by the loss of the ability to transport glucose into the cells of the body, because of either a lack insulin production or diminished insulin response. In a healthy person, minute elevations in blood glucose stimulate the production and secretion of insulin, the role of which is to increase glucose uptake into cells, returning the blood glucose to the optimal level. Insulin stimulates liver and skeletal muscle cells to take up glucose from the blood and convert it into glycogen, an energy storage molecule. It also stimulates skeletal muscle fibers to take up amino acids from the blood and convert them into protein, and it acts on adipose (fat) cells to stimulate the synthesis of fat. In diabetes, glucose saturates the blood stream, but it cannot be transported into the cells where it is needed and utilized. As a result, the cells of the body are starved of needed energy, which leads to the wasted appearance of many patients with poorly controlled insulin-dependent diabetes.
Prior to the discovery of insulin and its use as a treatment for diabetes, the only available treatment was starvation followed predictably by death. Death still occurs today with insulin treatment from over dosage of insulin, which results in extreme hypoglycemia and coma followed by death unless reversed by someone who can quickly get glucose into the patient. Also, death still occurs from major under dosage of insulin, which leads to hyperglycemia and ketoacidosis that can result in coma and death if not properly and urgently treated.
While diabetes is not commonly a fatal disease thanks to the treatments available to diabetics today, none of the standard treatments can replace the body's minute-to-minute production of insulin and precise control of glucose metabolism. Therefore, the average blood glucose levels in diabetics generally remain too high. The chronically elevated blood glucose levels cause a number of long-term complications. Diabetes is the leading cause of new blindness, renal failure, premature development of heart disease or stroke, gangrene and amputation, and impotence. It decreases the sufferer's overall life expectancy by one to two decades.
Diabetes mellitus is one of the most common chronic diseases in the world. In the United States, diabetes affects approximately 16 million people—more than 12% of the adult population over 45. The number of new cases is increasing by about 150,000 per year. In addition to those with clinical diabetes, there are approximately 20 million people showing symptoms of abnormal glucose tolerance. These people are borderline diabetics, midway between those who are normal and those who are clearly diabetic. Many of them will develop diabetes in time and some estimates of the potential number of diabetics are as high as 36 million or 25-30% of the adult population over 45 years.
Diabetes and its complications have a major socioeconomic impact on modem society. Of the approximately $700 billion dollars spent on healthcare in the US today, roughly $100 billion is spent to treat diabetes and its complications. Since the incidence of diabetes is rising, the costs of diabetes care will occupy an ever-increasing fraction of total healthcare expenditures unless steps are taken promptly to meet the challenge. The medical, emotional and financial toll of diabetes is enormous, and increase as the numbers of those suffering from diabetes grows.
Diabetes mellitus can be subdivided into two distinct types: Type 1 diabetes and Type 2 diabetes. Type 1 diabetes is characterized by little or no circulating insulin, and it most commonly appears in childhood or early adolescence. There is a genetic predisposition for Type 1 diabetes. It is caused by the destruction of the insulin-producing beta cells in the islets of Langerhans; which are scattered throughout the pancreas, an elongated gland located transversely behind the stomach. The beta cells are attacked by an autoimmune reaction initiated by some as yet unidentified environmental event. Possibly a viral infection or noninfectious agent (a toxin or a food) triggers the immune system to react to and destroy the patient's beta cells in the pancreas. The pathogenic sequence of events leading to Type 1 diabetes is thought to consist of several steps. First, it is believed that genetic susceptibility is an underlying requirement for the initiation of the pathogenic process. Secondly, an environmental insult mediated by a virus or noninfectious pathogen in food triggers the third step, the inflammatory response in the pancreatic islets (insulitis). The fourth step is an alteration or transformation of the beta cells such that they are no longer recognized as “self” by the immune system, but rather seen as foreign cells or “nonself”. The last step is the development of a full-blown immune response directed against the “targeted” beta cells, during which cell-mediated immune mechanisms cooperate with cytotoxic antibodies in the destruction of the insulin-producing beta cells. Despite this immune attack, for a period, the production of new beta cells is fast enough to stay ahead of the destruction by the immune system and a sufficient number of beta cells are present to control blood glucose levels. However, the number of beta cells gradually declines. When the number of beta cells drops to a critical level (10% of normal), blood glucose levels no longer can be controlled and progression to total insulin production failure is almost inevitable. It is thought that the regeneration of beta cells continues for a few years, even after functional insulin production ceases, but that the cells are destroyed as they develop to maturity.
To reduce their susceptibility to both the acute and chronic complications of diabetes, people with Type 1 diabetes must take multiple insulin injections daily and test their blood sugar multiple times per day by pricking their fingers for blood. They then have to decide how much insulin to take based on the food eaten and level of physical activity, amount of stress, and existence of any illness over the next few hours. The multiple daily injections of insulin do not adequately mimic the body's minute-to-minute production of insulin and precise control of glucose metabolism. Blood sugar levels are usually higher than normal, causing complications that include blindness, heart attack, kidney failure, stroke, nerve damage, and amputations. Even with insulin, the average life expectancy of a diabetic is 15-20 years less than a healthy person.
Type 2 diabetes usually appears in middle age or later, and particularly affects those who are overweight. Over the past few years, however, the incidence of Type 2 diabetes mellitus in young adults has increased dramatically. In the last several years, the age of onset for Type 2 diabetes in obese people has dropped from 40 years to 30 years. These are the new younger victims of this disease. In Type 2 diabetes, the body's cells that normally require insulin lose their sensitivity and fail to respond to insulin normally. This insulin resistance may be overcome for many years by extra insulin production by the pancreatic beta cells. Eventually, however, the beta cells are gradually exhausted because they have to produce large amounts of excess insulin due to the elevated blood glucose levels. Ultimately, the overworked beta cells die and insulin secretion fails, bringing with it a concomitant rise in blood glucose to sufficient levels that it can only be controlled by exogenous insulin injections. High blood pressure and abnormal cholesterol levels usually accompany Type 2 diabetes. These conditions, together with high blood sugar, increase the risk of heart attack, stroke, and circulatory blockages in the legs leading to amputation. Drugs to treat Type 2 diabetes include some that act to reduce glucose absorption from the gut or glucose production by the liver, others that reduce the formation of more glucose by the liver and muscle cells, and others that stimulate the beta cells directly to produce more insulin. However, high levels of glucose are toxic to beta cells, causing a progressive decline of function and cell death. Consequently, many patients with Type 2 diabetes eventually need exogenous insulin.
Another form of diabetes is called Maturity Onset Diabetes of the Young (MODY). This form of diabetes is due to one of several genetic errors in insulin-producing cells that restrict their ability to process the glucose that enters via special glucose receptors. Beta cells in patients with MODY cannot produce insulin correctly in response to glucose, which results in hyperglycemia. The patients treatment eventually leads to the requirement for insulin injections.
The currently available medical treatments for insulin-dependent diabetes are limited to insulin administration and pancreas transplantation with either whole pancreata or pancreatic segments.
Insulin therapy is by far more prevalent than pancreas transplantation. Insulin administration is conventionally either by a few blood glucose measurements and subcutaneous injections, intensively by multiple blood glucose measurements and through multiple subcutaneous injections of insulin, or by continuous subcutaneous injections of insulin with a pump. Conventional insulin therapy involves the administration of one or two injections a day of intermediate-acting insulin with or without the addition of small amounts of regular insulin. The intensive insulin therapy involves multiple administration of intermediate- or long-acting insulin throughout the day together with regular or short-acting insulin prior to each meal. Continuous subcutaneous insulin infusion involves the use of a small battery-driven pump that delivers insulin subcutaneously to the abdominal wall, usually through a 27-gauge butterfly needle. This treatment modality has insulin delivered at a basal rate continuously throughout the day and night, with increased rates programmed prior to meals. In each of these methods, the patient is required to frequently monitor his or her blood glucose levels and, if necessary, adjust the insulin dose. However, controlling blood sugar is not simple. Despite rigorous attention to maintaining a healthy diet, exercise regimen, and always injecting the proper amount of insulin, many other factors can adversely affect a person's blood-sugar including stress, hormonal changes, periods of growth, illness, infection and fatigue. People with Type 1 diabetes must constantly be prepared for life threatening hypoglycemic (low blood sugar) and hyperglycemic (high blood sugar) reactions. Insulin-dependent diabetes is a life threatening disease, which requires never-ending vigilance.
In contrast to insulin administration, whole pancreas transplantation or transplantation of segments of the pancreas is known to eliminate the elevated glucose values by regulating insulin release from the new pancreas in diabetic patients. Histologically, the pancreas is composed of three types of functional cells; a) exocrine cells that secrete their enzymes into a small duct, b) ductal cells that carry the enzymes to the gut, and c) endocrine cells that secrete their hormones into the bloodstream. The exocrine portion is organized into numerous small glands (acini) containing columnar to pyramidal epithelial cells known as acinar cells. Acinar cells comprise approximately 80% of the pancreatic cells and secrete into the pancreatic duct system digestive enzymes, such as, amylases, lipases, phospholipases, trypsin, chymotrypsin, aminopeptidases, elastase and various other proteins. Approximately 1.5 and 3 liters of alkaline fluid are released per day into the common bile duct to aid digestion.
The pancreatic duct system consists of an intricate, tributary-like network of interconnecting ducts that drain each secretory acinus, draining into progressively larger ducts, and ultimately draining into the main pancreatic duct. The lining epithelium of the pancreatic duct system consists of duct cells. Approximately 10% of the pancreas cells is duct cells. Duct cell morphology ranges from cuboidal in the fine radicles draining the secretory acini to tall, columnar, mucus secreting cells in the main ductal system.
Hormone producing islets are scattered throughout the pancreas and secrete their hormones into the bloodstream, rather than ducts. Islets are richly vascularized. Islets comprise only 1-2% of the pancreas, but receive about 10 to 15% of the pancreatic blood flow. There are three major cell types in the islets, each of which produces a different endocrine product: alpha cells secrete the hormone glucagon (glucose release); beta cells produce insulin (glucose use and storage) and are the most abundant of the islet cells; and delta cells secrete the hormone somatostatin (inhibits release of other hormones). These cell types are not randomly distributed within an islet. The beta cells are located in the central portion of the islet and are surrounded by an outer layer of alpha and delta cells. Besides insulin, glucagon and somatostatin, gastrin and Vasoactive Intestinal Peptide (VIP) have been identified as products of pancreatic islets cells.
Pancreas transplantation is usually only performed when kidney transplantation is required, which makes pancreas-only transplantations relatively infrequent operations. Although pancreas transplants are very successful in helping people with insulin-dependent diabetes improve their blood sugar control without the need for insulin injections and reduce their long-term complications, there are a number of drawbacks to whole pancreas transplants. Most importantly, getting a pancreas transplant involves a major operation and requires the use of life-long immune suppressant drugs to prevent the body's immune system from destroying the pancreas. The pancreas is destroyed in a manner of days without these drugs. Some risks in taking these immuno-suppressive drugs are the increased incidence of infections and tumors that can be life threatening in their own right. The risks inherent in the operative procedure, the requirement for life-long immunosuppression of the patient to prevent rejection of the transplant, and the morbidity and mortality rate associated with this invasive procedure, illustrate the serious disadvantages associated with whole pancreas transplantation for the treatment of diabetes. Thus, an alternative to insulin injections or pancreas transplantation would fulfill a great public health need.
Islet transplants are much simpler (and safer) procedures than whole pancreas transplants and can achieve the same effect by replacing the destroyed beta cells. As discussed above, when there are insufficient numbers of beta cells, or insufficient insulin secretion, regardless of the underlying reason, diabetes results. Reconstituting the islet beta cells in a diabetic patient to a number sufficient to restore normal glucose-responsive insulin production would solve the problems associated with both insulin injection and major organ transplantation. Microencapsulation and implantation of islet cells into diabetic patients holds promise for treatment of those with diabetes.
Encapsulation of cells for the potential of treating a number of diseases and disorders has been discussed in the literature. The concept was suggested as early as 100 years ago, but little work was done prior to the 1950's when immunologists began using encapsulated cells with membrane devices to separate the cells from the host to better understand the different aspects of the immune system. Research on implantation was underway in the 1970's and 1980's with the first review written in 1984. Several additional reviews have been written since then explaining the different approaches and types of devices under development. Cell encapsulation technology has potential applications in many areas of medicine. For example, some important potential applications are treatment of diabetes (Goosen, M. F. A., et al. (1985) Biotechnology and Bioengineering, 27:146), production of biologically important chemicals (Omata, T., et al. (1979) “Transformation of Steroids by Gel-Entrapped Nacardia rhodocrous Cells in Organic Solvent” Eur. J. Appl. Microbiol. Biotechnol. 8:143-155), and evaluation of anti-human immunodeficiency virus drugs (McMahon, J., et al. (1990) J. Nat. Cancer Inst., 82(22) 1761-1765).
There are three main types of encapsulated devices, which can best be categorized by describing the form of encapsulation. The three categories are a] macrodevices, b] microcapsules, and c] conformal coatings.
Macrodevices are larger devices containing membranes in the form of sheets or tubes for permselectivity and usually supporting structures. They contain one or several compartments for the encapsulated cells. They are designed for implantation into extravascular or vascular sites. Some are designed to grow into the host to increase oxygen diffusion into these large devices. Others are designed to have no reaction by the host, thus increasing their ease of removal from different sites. There have been two major types of macrodevices developed: a] flat sheet and b] hollow fiber.
Among the flat sheet devices, one type (Baxter, Theracyte) is made of several layers for strength and has diffusion membranes between support structures with loading ports for replacing the cells. The other type is more simple in design. The device uses alginate based membranes and other supporting membranes to encapsulate islets within an alginate matrix between the sheets. The complex device is designed to grow into the body to increase diffusion of oxygen. Due to its relatively large size, there are few sites in the body able to accommodate it for the treatment of a disease like diabetes. Since it grows into the body and the contained cells are not expected to survive for more than a few years, multiple cell removals and reloading of new cells is required for the long-term application of this device. It has proven quite difficult to flush and reload this type of device while at the same time maintaining the critical cell compartment distance for oxygen diffusion.
The second flat sheet style of device is designed to be an “all in/all out” device with little interaction with the host. For the diabetes product, it has been quite difficult to place this device into the intraperitoneal cavity of large animals, while maintaining its integrity. This has been due to the difficulty in securing it in the abdomen so that the intestines cannot cause it to move or wrinkle, which may damage or break the device.
The other major macrodevice type is the hollow fiber, made by extruding thermoplastic materials into hollow fibers. These hollow fibers can be made large enough to act as blood conduits. One model is designed to be fastened into the host's large blood vessels and the encapsulated cells are behind a permselective membrane within the device. This type has shown efficacy in large animal diabetic trials, but has been plagued by problems in the access to the vascular site. Both thrombosis and hemorrhage have complicated the development of this approach with it currently being abandoned as a clinically relevant product. Another model using hollow fibers is much smaller in diameter and designed to be used as an extravascular device. Due to low packing densities, the required cell mass for encapsulation causes the length of this type of hollow to approach many meters. Therefore, this approach was abandoned for treating diabetes since it was not clinically relevant. In addition, sealing the open ends of the fiber is not trivial and strength has been a problem depending upon the extravascular site.
The microcapsule was one of the first to offer potential clinical efficacy. Alginate microcapsules were used to encapsulate islets, which eliminated diabetes in rodents when implanted intraperitoneally. However, nearly 25 years have passed since these first reports without the ability to demonstrate clinical efficacy. One of the problems associated with microcapsules is their relatively large size in combination with low packing densities of cells, especially for the treatment of diabetes. Another is the use of alginate; an ionically crosslinked hydrogel dependent upon the calcium concentration for its degree of crosslinking. The permselectivity of pure alginate capsules has been difficult to control with the vast majority being wide open in terms of molecular weight cutoff. Varieties of positively charged crosslinked agents, such as polylysine, have been added as a second coating to provide permselectivity to the capsule. However, polylysine and most other similar molecules invite an inflammatory reaction requiring an additional third coating of alginate to reduce the host's response to the capsule. In addition, it has been difficult to produce very pure alginates that are not reactive within the host after implantation. Trying to reduce the size of the alginate microcapsules causes two major problems. First, the production of very large quantities of empty capsules without any cells. Second, the formation of smaller capsules results in poorly coated cells. There is no force to keep the contained cells within the center of the microcapsule, which causes the risk of incomplete coatings to go up exponentially with the decrease in the size of the capsules. Production of conformal coatings has not been demonstrated with alginate microcapsules.
The last category of cell encapsulation is conformal coating. A conformally coated cell aggregate is one that has a substantially uniform cell coating around the cell aggregate regardless of size or shape of the aggregate. This coating not only may be uniform in thickness, but it also may be uniform in the protective permselective nature of the coating that provides uniform immune protection. Furthermore, it may be uniform in strength and stability, thus preventing the coated material from being violated by the host's immune system.
An important aspect to the feasibility of using these various methods is the relevant size and implant site needed to obtain a physiological result of 15,000 IEQ/kg-BW. Injecting isolated islets into the Portal Vein requires 2-3 ml of pack cells. A macro-device consisting of a flat sheet that is 1 islet thick (˜500 μm) requires a surface area equivalent to 2 US dollar bills. A macro-device consisting of hollow fibers with a loading density of 5% would need 30 meters of fiber. Alginate microcapsules with an average diameter of 400-600 μm would need a volume of 50-170 ml. However, PEG conformal coating of islets which produces a 25-50 μm thick covering would only need a volume of 6-12 ml and could be injected into almost any area in the body.
The stringent requirements of encapsulating polymers for biocompatibility, chemical stability, immunoprotection and resistance to cellular overgrowth restrict the applicability of prior art methods of encapsulating cells and other biological materials. The membranes must be non-toxically produced in the presence of cells, with the qualities of being permselective, chemically stable, and very highly biocompatible.
Synthetic or natural materials intended to be exposed to biological fluids or tissues are broadly classified as biomaterials. These biomaterials are considered biocompatible if they produce a minimal or no adverse response in the body. For many uses of biomaterials, it is desirable that the interaction between the physiological environment and the material be minimized. For these uses, the material is considered “biocompatible” if there is minimal cellular growth on its surface subsequent to implantation, minimal inflammatory reaction, and no evidence of anaphylaxis during use. Thus, the material should neither elicit a specific humoral or cellular immune response nor a nonspecific foreign body response.
Materials that are successful in preventing all of the above responses are relatively rare. Biocompatibility is more a matter of degree rather than an absolute state. The first event occurring at the interface of any implant with surrounding biological fluids is protein adsorption (Andrade, J. D. et al. (1986) V. Adv. Polym. Sci., 79:1-63). In the case of materials of natural origin, it is conceivable that specific antibodies for that material exist in the repertoire of the immune defense mechanism of the host. In this case, a strong immune response can result. Most synthetic materials, however, do not elicit such a reaction. They can either activate the complement cascade and/or adsorb serum proteins, such as, cell adhesion molecules (CAMs), which mediate cell adhesion (Buck, C. A. et al. (1987) Ann. Rev. Cell Biol., 3:179-205).
Proteins can adsorb on almost any type of material. They have regions that are positively and/or negatively charged, as well as, hydrophilic and hydrophobic. Thus, they can interact with implanted material through any of these various regions, resulting in cellular proliferation at the implant surface. Complement fragments such as C3b can be immobilized on the implant surface and act as chemoattractants. They in turn can activate inflammatory cells, such as macrophages and neutrophils, and cause their adherence and activation on the implant. These cells attempt to degrade and digest the foreign material.
In the event that the implant is nondegradable and is too large to be ingested by large single activated macrophages, the inflammatory cells may undergo frustrated phagocytosis. Several such cells can combine to form foreign body giant cells. In this process, these cells release peroxides, hydrolytic enzymes, and chemoattractant and anaphylactic agents such as interleukins, which increase the severity of the reaction. They also induce the proliferation of fibroblasts on foreign surfaces.
Past approaches to enhancing biocompatibility of materials started with attempts at minimization of interfacial energy between the material and its aqueous surroundings. Similar interfacial tensions of the solid and liquid were expected to minimize the driving force for protein adsorption and this was expected to lead to reduced cell adhesion and thrombogenicity of the surface. For example, Amudeshwari et al. used collagen gels crosslinked in the presence of HEMA and MMA (Amudeswari, S., et al. (1986) J. Biomed. Mater. Res. 20:1103-1109). Desai and Hubbell showed a poly(HEMA)-MMA copolymer to be somewhat non-thrombogenic (Desai, N. P. et al. (1989) J. Biomaterials Sci., Polym. Ed., 1:123-146; Desai, N. P. et al. (1989) Polym. Materials Sci. Eng., 62:731).
Hubbell et al. (U.S. Pat. No. 5,529,914 and related patents) disclose methods for the formation of biocompatible membranes around biological materials using photopolymerization of water-soluble molecules. Each of these methods utilizes a polymerization system containing water-soluble macromers, polymerization using a photoinitiator (such as a dye), and radiation in the form of visible or long wavelength UV light.
Due to the inability of those of skill in the art to provide one or more important properties of successful cell encapsulation, none of the encapsulation technologies developed in the past have resulted in a clinical product. These properties can be broken down into the following categories:
Biocompatibility—The materials used to make an encapsulating device must not elicit a host response, which may cause a non-specific activation of the immune system by these materials alone. When considering immunoisolation, one must recognize that it will only work in the situation where there is no activation of the host immune cells to the materials. If there is activation of the host immune cells by the materials, then the responding immune cells will surround the device and attempt to destroy it. This process produces many cytokines that will certainly diffuse through the capsule and most likely destroy the encapsulated cells. Most devices tested to date have failed in part by their lack of biocompatibility in the host.
Permselectivity—There exists an important balance between having the largest pores as possible in the capsule surrounding the encapsulated cells to permit all the nutrients and waste products to pass through the capsule to permit optimal survival and function, while at the same time, the smallest pore size as possible in the capsule to keep all elements of the immune system away from the encapsulated cells to prevent degradation of the cells. Small pores capable of keeping out immune cytokines also cause the death of the encapsulated cells from a lack of diffusion of nutritional elements and waste products. The optimal cell encapsulation has an exact and consistent permselectivity, which allows maximal cell survival and function, as well as, provides isolation from the host immune response. Ideally, this encapsulation technology should offer the ability to select and change the pore size as required by the encapsulated cells and their function, as well as pore size variation based on whether the cells are allograft or xenograft cells.
Encapsulated Cell Viability and Function—The encapsulating materials should not exhibit cytotoxicity to the encapsulated cells either during the formation of the coatings or on an ongoing basis, otherwise the number of encapsulated cells will decrease and risk falling short of the number required for a therapeutically effective treatment of a disease or disorder.
Relevant Size—Many devices are of such a large size that the number of practical implantation sites in the host is limited. Another factor is the relative diffusion distance between the encapsulated cells and the host. The most critical diffusive agent for cell survival is oxygen. These diffusion distances should be minimal since the starting partial pressure of oxygen is in the range of 30-40 mm Hg at the tissue level in the body. There is little tolerance for a reduction in diffusive distances, due to the initially low oxygen partial pressure. This would further lower the oxygen concentration to a point where the cells cannot adequately function or survive.
Cell Retrieval or Replacement—The encapsulating device should be retrievable, refillable, or biodegradable, allowing for replacement or replenishment of the cells. Many device designs have not considered the fact that encapsulated cells have a limited lifetime in the host and require regular replacement.
Therapeutic Effect—The implant should contain sufficient numbers of functional cells to have a therapeutic effect for the disease application in the host.
Clinical Relevance—The encapsulating cell device should have a total volume or size that allows it to be implanted in the least invasive or most physiologic site for function, which has a risk/benefit ratio below that faced by the host with the current disease or disorder.
Commercial Relevance—The encapsulating cell device should be able to meet the above requirements in order for it to be produced on an ongoing basis for the long-term treatment of the disease process for which it has been designed.
All of the above factors must be taken into consideration when evaluating a specific technique, method or product for use in implantation of islets to alleviate the effects of diabetes.
Transplantation of human islets with immunosuppression is done by injecting unencapsulated islets into the portal vein by direct injection percutaneously between the ribs, into the liver, and then the portal vein by fluoroscopic direction. Essentially all of the human islet transplants have been done by this technique, except for the first ones done by umbilical vein injection via a cutdown. A major risk of this procedure is the fact that injection of islets into the portal vein leads to increased portal venous pressures depending on the rate of infusion and the amount infused. Another risk has been elevated portal venous pressures from large volumes of injected islet tissues that are not sufficiently purified. This also leads to portal venous thrombosis as a complication of this procedure. As the interventional radiologist prepares to withdraw the catheter, a bolus of gelatin is left behind to prevent hemorrhaging from the injection site. Unfortunately, several patients have had bleeding episodes following this procedure.
In addition to injecting the islets into the portal vein, a few patients have had their islets injected into the body of the spleen. The spleen is more fragile than the liver so these injections were performed at the time of kidney transplantation at which time the splenic injection could be done as an open procedure. Freely injecting the islets into the peritoneal cavity has been performed in mouse transplants without difficulty. In using this site in larger animals or humans, it has been found that twice the number of islets is needed in the peritoneal cavity than required in the portal vein implants. If any rejection or inflammatory reactions occur, then adhesions tend to form between the loops of intestine, as well as, to the omentum. This reaction can lead to additional problems long term, such as, bowel obstruction. Thus, the ability to perform encapsulated islet implants into the subcutaneous site would significantly reduce the complications associated with these other sites.
Attempts at subcutaneous implantation of encapsulated islets have been unable to produce sustainable results in the treatment of diabetes, probably due to some or all of the scientific challenges described above. Tatarkiewicz et al. (Transplantation Proceedings 1998, 30, 479-480) discloses the implantation of rat islets, enclosed in tissue diffusion ported devices, subcutaneously in mice. Kawakami et al. (Cell Transplantation 1997 6, 5:541-545) implanted pancreatic beta cells, encapsulated in agarose-PSSa, subcutaneously in rats. Insulin secretion from the cells was maintained after transplantation. However, this study only examined subcutaneous implantation of the encapsulated islet cells over a one-week period. No evidence has been provided that the insulin secretion response of the cells could be maintained long term in a subcutaneous implant. Kawakami et al. (Transplantation 2002, 73,122-129) enclosed rat islets in an agarose/poly(styrene sulfonic acid) mixed gel and implanted the encapsulated cells into a prevascularized subcutaneous site. Stockley et al. (J. Lab. Clin. Med. 135:484-492) encapsulated allogenic MDCK cells engineered to secrete human growth hormone in alginate-poly-L-lysine-alginate and implanted them subcutaneously. The encapsulated cells of Stockley et al. can be estimated as having a diameter of approximately 1.5 mm, if it is assumed that the capsule volume used is 100 μl and this volume does not comprise components other than the encapsulated cells. Stockley provides no information about the actual volume of encapsulated cells that are applied. One of skill in the art would be unable to determine the desired volume of encapsulated cells needed to administer to a subject.